Blood Cell Production and Function
A sensitised genetic screen in mice reveals that the Ets family transcription factor Erg regulates haemopoietic stem cells
SJ Loughran, EA Kruse, CD Hyland, TA Willson, D Metcalf, WS Alexander in collaboration with D Hacking, CA de Graaf, KJ Henley, AK Voss, DJ Hilton, BT Kile (Molecular Medicine Division), S Ellis (Peter MacCallum Cancer Centre, Melbourne) Pub ref: 93
The Ets related gene (Erg) is a transcription factor that has been strongly implicated in cancer. ERG is rearranged in rare cases of human myeloid leukaemia and Ewing sarcoma and its overexpression is a poor prognostic indicator for patients with acute myeloid leukaemia with a normal karyotype. The recent demonstration that chromosomal rearrangements place ERG under the control of androgen-response elements in more than half of all prostate cancers provides further compelling evidence that deregulation of Erg contributes to malignancy. Despite mounting evidence for a role of ERG in cancer, the physiological role of Erg has remained obscure. Mice with a germline mutation in Erg were isolated from a sensitised ENU mutagenesis screen for regulators of haemopoiesis, allowing for the first time, analysis of the indispensable functions of Erg in vivo.
The multilineage defect 2 (Mld2) pedigree was established from a first generation offspring of ENU-injected Mpl-/- mice that displayed low numbers of red and white blood cells as well as exacerbation of the thrombocytopenia typical of Mpl-deficient animals. The ENU-induced mutation was mapped to chromosome 16 and shown to be a mutation predicted to cause substitution of proline for serine at amino acid 329 in Erg. Although retaining DNA binding capacity, the ErgMld2 protein exhibited defective transactivation ability and appeared biologically inert. The physiological role of Erg was dissected in Mld2 mice on a wild-type Mpl+/+ background. In contrast to the Mpl-/- background, Erg+/Mld2 mice on a wild-type background were not anemic, but were mildly leucopenic and thrombocytopenic. Lineage committed haemopoietic progenitor cells of all lineages were reduced in Erg+/Mld2 mice and transplantation assays established severe defects in the capacity of Erg+/Mld2 haemopoietic stem cells to compete with wild-type cells in reconstitution of the blood-forming system in irradiated normal recipients. Flow cytometric phenotyping revealed an imbalance in the relative numbers of long-term and short-term haemopoietic stem cells suggesting Erg is critical for haemopoietic stem cell function and may have a particular role in the maintenance of specific cells within the stem cell pool.
Mice homozygous for the ErgMld2 allele failed to survive embryogenesis beyond E11.5. Unlike mice lacking Fli1, a close relative of Erg, ErgMld2/Mld2 embryos exhibited no evidence of disrupted vasculogenesis or primitive haemopoiesis but displayed reduced blood cell development consistent with a failure of definitive haemopoiesis. Transcriptional profiling in haemopoietic stem cell populations by microarray revealed that expression of most genes was unaltered with only a handful of transcripts significantly up- or down-regulated in Erg+/Mld2 cells. None of the genes showing altered transcription have previously been implicated in haemopoietic stem cell biology, raising the exciting prospect that Erg regulates a key set of novel genes in haemopoiesis.
Sections of yolk sac blood islands at E8.5 (top) and primitive erythrocytes in the embryonic dorsal aorta at E10.5 (bottom), showing absence of major defects in mice homozygous for the ErgMld2 allele (ErgMld2/Mld2)
Suppression of Mpl-/- thrombocytopenia by mutation of the gene encoding p300 in Plt6 mice
M Kauppi, CD Hyland, D Metcalf, AA Hilton, NA Nicola, WS Alexander in collaboration with JM Murphy, CA de Graaf, KT Greig, BT Kile, DJ Hilton (Molecular Medicine Division)
Plt6 mice, which have a mutation in the gene encoding the transcriptional co-regulator p300, emerged from an ENU mutagenesis screen for suppressors of thrombocytopenia in Mpl-/- mice. The Plt6 mutation causes disrupted interaction between p300 and its partner protein c-Myb, resulting in mild amelioration of thrombocytopenia in Mpl-/- p300Plt6/+ heterozygotes and supraphysiological levels of circulating platelets in homozygous mice. General expansion in megakaryocytopoiesis accompanied the increased platelet numbers in Plt6 mice and was reminiscent of the phenotype observed in mice with mutations affecting c-Myb. Thus, the c-Myb/p300 transcriptional regulatory complex is a key regulator of megakaryocyte development, down-regulation of which amplifies TPO-independent platelet production.
A kinase-like protein is required for regulation of megakaryocytopoiesis and haemopoietic stem cells
E Hatchell, IJ Majewski, D Stockwell, J-G Zhang, WS Alexander in collaboration with GM Tannahill, JM Murphy, DJ Hilton (Molecular Medicine Division)
A mutation causing increased circulating platelet count was isolated from an ENU mutagenesis screen. Mice carrying the mutation, which maps to a gene encoding an uncharacterised kinase-like protein, exhibit a mild increase in the number of megakaryocytes and their progenitors, consistent with elevated platelet counts occurring as the result of expanded cellular production. Intriguingly, the mutant mice also show signs of haemopoietic stem cell deficiency. Ongoing dissection of this phenotype will define the biological roles of this novel regulator, which appears to regulate blood cells at multiple levels.
Polycomb Repressive Complex 2 (PRC2) restricts blood stem cell function
IJ Majewski, EJ McManus, AA Hilton, CD Hyland, JE Corbin, D Metcalf, WS Alexander in collaboration with ME Blewitt, CA de Graaf, DJ Hilton (Molecular Medicine Division), M Bahlo, GK Smyth (Bioinformatics Division) Pub ref: 95
Polycomb group proteins are transcriptional repressors that play a central role in the establishment and maintenance of gene expression patterns during development. Rather than binding to DNA, Polycomb group proteins mediate transcriptional control via post-translational modifications made on histone tails. PRC2 catalyses tri-methylation of Histone H3 at Lysine 27; this modification serves as a signpost for the basal transcriptional machinery and identifies genes that should not be transcribed. Using mice with a mutation in Suz12, a key component of PRC2, we have shown that impairment of PRC2 boosts the activity of haemopoietic stem cells (HSCs). Mice that carry one functional copy of Suz12 develop normally, but HSCs isolated from these animals are more competitive than wild-type cells and make a greater contribution to haemopoiesis in transplantation experiments. Mutations in Eed or Ezh2, the other components of the complex, induce a similar phenotype, which suggests that HSCs are exquisitely sensitive to the dosage of the complex.
To investigate the molecular targets of the PRC2 complex in the HSC compartment, we examined changes in gene expression in cells deficient in Suz12. We identified a distinct set of genes regulated by Suz12 in haemopoietic cells, including eight genes that are highly responsive to PRC2 function within the progenitor compartment. These data suggest that PRC2 maintains a specific gene expression pattern in haemopoiesis that is indispensable to normal stem cell function. Future studies will explore the role of PRC2 in regulating stem cell self-renewal, proliferation and homing.
Polycomb group proteins are important regulators of body segmentation. A skeletal preparation demonstrates normal development in mice heterozygous for a null allele of Suz12 (left). The representation of Suz12-deficient blood cells increased steadily during serial transplantation (right), indicating increased HSC activity.
The Ets family transcription factor Erg regulates stem cell function
EA Kruse, DF Hacking, CA de Graaf, TA Willson, KJ Henley, AK Voss, DJ Hilton, BT Kile in collaboration with SJ Loughran, CD Hyland, D Metcalf, WS Alexander (Cancer and Haematology Division), S Ellis (Peter MacCallum Cancer Centre) Pub ref: 93
Members of the Ets family of transcription factors, such as PU.1, Tel, MEF and Fli-1, are key regulators of haemopoiesis: their characterisation has provided important insights into stem cell function and the formation of blood in health and disease. Indirect evidence implicated an additional family member, Erg, in these processes. The gene encoding Erg is rearranged in a number of human cancers, including Ewing’s sarcoma, acute myeloid leukaemia, and more than half of all prostate cancers. Despite being a known proto-oncogene, definitive proof of Erg’s normal physiological role has been elusive owing to the absence of in vivo models of Erg function. Using a genetic screen designed to identify molecular regulators of haemopoietic stem cell function in mice, we recently isolated the first mutant allele of Erg, an ENU-induced missense mutation in the DNA-binding domain, denoted ErgMld2. Animals homozygous for this mutation suffer a failure of definitive haemopoiesis and died at mid gestation. Mice carrying only one copy of ErgMld2 are viable as adults, but exhibit a significant reduction in haemopoietic stem cell number. In transplantation assays, both whole bone marrow and purified stem cells exhibited a profound engraftment defect, indicating that Erg is essential for the maintenance of stem cell number and for normal stem cell function. Microarray analyses detected expression changes in multiple genes, none of which were known stem cell regulators. This suggests Erg may control the transcription of novel pathways in haemopoietic stem cells. It also raises the possibility that rearrangements in the human Erg gene contribute to malignant transformation by conferring stem cell-like properties on normal cells. Mice carrying the ErgMld2 mutation thus represent a useful tool for studies designed to identify the normal targets of this critical transcription factor in blood cell development and cancer.
ErgMld2/Mld2 embryos exhibit essentially normal, but dilated vasculature, and show developmental delay. (A-B) Embryos at E10.5 following whole mount anti‑PECAM‑1 immunohistochemistry. Bars, 2 mm. (C-D) The cranial region of the same embryos. Bars, 500 µm.
The role of Bcl-2 proteins in platelet production and function
EC Josefsson, MW Goschnick, MJ White, KJ Henley, BT Kile in collaboration with KD Mason, DCS Huang (Molecular Genetics of Cancer Division), AW Roberts (Cancer and Haematology Division), SM Schoenwaelder, SP Jackson (Australian Centre for Blood Diseases)
Platelets are small anucleate cells essential for blood clotting. They are dependent on the Bcl-2 family protein Bcl-xL for survival. Bcl-xL restrains the activity of pro-death Bak and Bax, which initiate platelet apoptosis and clearance from the blood stream. We are studying mice lacking these proteins to determine how they contribute to other aspects of platelet function, such as phosphatidylserine exposure. We are also interested in whether they mediate platelet production by megakaryocytes, which have been reported to utilise components of the apoptotic machinery, such as caspases, during the process of platelet shedding.
The role of Erg and Fli-1 in haemopoiesis
EA Kruse, CL Carmichael, CA de Graaf, DJ Hilton, BT Kile in collaboration with SJ Loughran, A Ng, D Metcalf, WS Alexander (Cancer and Haematology Division)
The Ets family transcription factors Erg and Fli-1 regulate numerous aspects of haematopoiesis and are implicated in a broad range of human malignancies including leukaemia and prostate cancer. We are studying mice lacking Erg and Fli-1 in order to delineate their physiological functions and identify their transcriptional targets. Preliminary evidence suggests that although these two proteins are not functionally equivalent, they may co-regulate a set of genes in haemopoietic stem cells and megakaryocytes. The latter are extremely sensitive to perturbations in the Erg/Fli-1 complex and we are now elucidating its role in the development of the megakaryocyte lineage.
TPO independent megakaryocyte production in c-Myb
CA de Graaf, DJ Hilton in collaboration with CD Hyland, M Kauppi, WS Alexander (Cancer and Haematology Division), GK Smyth (Bioinformatics Division)
The chief cytokine that regulates megakaryocyte development is thrombopoietin (TPO), which signals through its receptor c-Mpl. c-Mpl-/- mice have a tenth of the platelets and megakaryocyte progenitors of wild-type mice. The Plt4 mutation in the transcription factor c-Myb suppresses this phenotype, resulting in platelet levels above wild-type, even in a c-Mpl-/- mouse. Microarrays on multipotent progenitor cell populations confirm that c-Mpl-/- and c-MybPlt4/Plt4 have opposing effects on known megakaryocyte genes. However, there is a larger group genes that they influence in the same direction. Further analysis of this group of genes may shed light on TPO independent pathways of megakaryocyte development.
A mouse recombination hotspot identifies a novel gene implicated in type 1 diabetes
IKL Tan, F Quirk, L Mackin, N Wang, TC Brodnicki in collaboration with K Shortman (Immunology Division), TW Kay (St Vincent’s Institute),
G Morahan (Western Australian Institute for Medical Research)
We have confirmed a diabetes susceptibility locus, termed Idd11, in diabetes-prone NOD mice. A panel of congenic NOD mouse strains, harbouring different C57BL/6-derived Chr4 intervals, have pinpointed a recombination hotspot that encompasses a sequence associated with diabetes. This sequence is located within a previously undescribed gene that is differentially expressed in immunological tissues between diabetes resistant and susceptible mouse strains. We have also observed that congenic mice, while protected from diabetes, still develop insulitis. Characterisation of this novel gene may provide insights regarding the regulation of autoreactive lymphocytes that mediate the destruction of insulin-producing β cells.
Characterising potential genetic interplay between autoimmunity and infectious disease
N Wang, TC Brodnicki in collaboration with GT Belz (Immunology Division), RA Strugnell, O Wijburg (University of Melbourne)
We have shown that diabetes-prone NOD mice harbouring a C57BL/6-derived Chr13 interval have an increased incidence of type 1 diabetes, confirming that a non-diabetes-prone mouse strain can harbour a diabetogenic allele. Interestingly, these congenic NOD mice are resistant to Listeria infection and exhibit a different immunological response compared to wild-type NOD mice. Given that the C57BL/6 allele is diabetogenic, it may be that this allele is maintained within the Mus species because it provides resistance to bacterial infection. We are currently characterising this congenic interval to determine if a single gene regulates a checkpoint between immunity and autoimmunity.
A mouse susceptibility locus moderates Sjögren’s syndrome, but not type 1 diabetes
RA Burt, L Watkins, F Quirk, TC Brodnicki in collaboration with P Morgan, J-G Zhang (Cancer and Haematology Division)
Genetic studies of the diabetes-prone NOD mouse have identified a region on Chr7 linked to type 1 diabetes and salivary gland inflammation, termed sialadenitis, which is a symptom of Sjögren’s syndrome. We established a congenic NOD mouse strain harbouring an NZW-derived Chr7 interval, which exhibits reduced sialadenitis, but still develops diabetes. Anomalous increases for anti-nuclear antibodies and marginal-zone B-cells were also identified in congenic mice, indicating that the NZW-derived Chr7 interval is a complex effect on the NOD immune system and most likely harbours more than one locus contributing to autoimmunity.
Management of mouse colony and phenotype data
J Choi, J Wettenhall
Automated blood cell counts, flow cytometry data and other arbitrary measurements constitute elaborate phenotypes that can be used to screen progeny of ENU-injected mice. An Animal Management Application has been developed to combine the flexibility of a spreadsheet-based system for storing complex phenotypes with the quality control of a strict mouse colony database system. This facilitates straightforward management of breeding, ethics, genotype and phenotype data. The Animal Management Application uses a system that shows the state of each mouse - alive, discarded, currently mating - and indicates to the animal technicians which mice require tasks to be completed, as requested by scientists.
Discovery of diabetes genes using transposon mutagenesis in the NOD mouse
CM Elso, RA Burt, F Quirk, L Mackin, TC Brodnicki in collaboration with SJ Foote (Menzies Research Institute, Hobart)
Rather than identifying polymorphisms associated with type 1 diabetes, a more clinically relevant strategy is to identify genes which, when disrupted, prevent disease. Such genes represent promising therapeutic targets. We have introduced the Sleeping Beauty transposon into the diabetes-prone NOD mouse strain. This transposon can jump around the mouse genome and generate mutations that can be screened for their effect upon disease in NOD mice. The transposon also acts as a DNA tag for rapid identification of disrupted genes. In this manner, we aim to prioritise the characterisation of transposon mutations and accelerate discovery of genes that affect diabetes onset.
Aire controls the expression of thousands of genes in thymic medullary epithelial cells
SA Kinkel, FX Hubert, PE Crewther, HS Scott in collaboration with GM Davey, WR Heath (Immunology Division), B Phipson, GK Smyth (Bioinformatics Division), KE Webster (Garvan Institute of Medical Research)
Effective deletion of autoreactive thymocytes is necessary to establish central tolerance and protect from autoimmune disease. Medullary thymic epithelial cells (mTEC) play a unique role in this process by promiscuously expressing a range of tissue-specific antigens (TSAs) that are presented to developing thymocytes. Aire is currently the only transcription factor known to control this process. We have performed gene expression arrays on mTEC from wild-type and Aire-/- mice and shown that Aire has thousands of target genes, including TSAs, chemokines and cytokines important in antigen presentation, cell migration and cell interactions. We are currently performing experiments to identify whether Aire binds directly to target gene promoters and what other proteins are involved in controlling promiscuous expression.